Fungal pathogenesis: Combatting the oxidative burst

Plants respond to microbial attack with a lethal burst of reactive oxygen species. How then, do pathogens successfully invade plants? Unexpectedly, a link between primary metabolism and suppression of plant immunity allows the rice blast fungus Magnaporthe oryzae to grow in such a hostile environment.

Plant-infecting fungi and oomycetes are an ever present threat to global food security — each year they destroy sufficient food to feed half a billion people1. Understanding how these pathogens infect and colonize host plants is therefore crucial if we are to develop new strategies to fight fungal diseases and improve plant health. The rice blast fungus Magnaporthe oryzae is responsible for the most devastating disease of cultivated rice, the primary staple for more than half of the world's population2. Strains of the same fungus also cause blast disease of wheat, a new disease that is currently causing a severe outbreak in Bangladesh and India3. Because of its economic significance and genetic tractability, rice blast disease has also emerged as a major model for studying the molecular and cellular basis of fungal infection2,4. Similar to many pathogenic fungi, M. oryzae has evolved highly sophisticated ways to invade plant cells and disable their defence systems.

One of the earliest responses of plants to microbial attack is the induction of a rapid, transient burst of reactive oxygen species5 (ROS). This oxidative burst is a potent defence reaction that will kill any unsuspecting microorganism. How can pathogens still successfully invade plant cells and contend with such high concentrations of ROS? In this issue of Nature Microbiology, Marroquin-Guzman and colleagues report that a link between primary fungal metabolism and the suppression of plant immunity enables M. oryzae to protect itself from nitrooxidative stress and maintain redox balance within living rice cells6, thereby facilitating its growth within such a hostile environment.

To initiate rice infection, a fungal spore adheres tightly to the rice leaf surface, germinates, and rapidly elaborates a specialized infection structure called an appressorium that is used to breach the tough outer leaf cuticle4. Once inside a host cell, the fungus develops bulbous, branched hyphae that maintain intimate contact with the plasma membrane of the living plant cell, forming a specialized biotrophic interfacial complex7. The pathogen and host then engage in an intense molecular dialogue; plant metabolism is reprogrammed to the pathogen's benefit and the host immune response is suppressed4,7. Much recent research has focused on fungal effector proteins, a highly diverse group of secreted molecules that target immune responses in the host to facilitate pathogen infection and spread8. However, fungi have clearly evolved additional ways to keep plant defences at bay. Marroquin-Guzman and colleagues identified and characterized nitronate monooxygenase (NMO) from M. oryzae, an enzyme normally used by fungal cells to protect themselves from nitrooxidative stress6. They show, however, that NMO is also required for utilization of nitrate and nitrite as nitrogen sources and for maintaining redox balance within living rice cells during pathogen colonization. The latter role of NMO is essential for pathogenicity, because it provides a novel mechanism by which the fungus can prevent elicitation of the plant oxidative burst (Fig. 1).

Figure 1: The Magnaporthe oryzae response to oxidative insults.
figure1

M. oryzae uses the nitrooxidative stress response enzyme Nmo to maintain cell redox balance in infected rice cells and prevent the host defence response. a, Nmo2 is required for mitigating RNS-induced lipid damage during growth of M. oryzae on nitrate (NO3) and nitrite (NO2). Oxidative denitrification by Nmo2 protects the fungal cell against nitroalkanes generated as a result of nitrooxidative stress during NO3 and NO2 metabolism. b, Expression of fungal Nmo2 is upregulated during appressorium-mediated infection of rice plants and prevents the accumulation of ROS through an unknown mechanism, thereby maintaining redox homeostasis and allowing host colonization by the pathogen (left). Loss of Nmo2 results in accumulation of ROS in infected rice cells, triggering an oxidative burst that activates plant innate immunity and blocks fungal infection (right).

NMO enzymes were described in fungi more than 60 years ago, and their primary physiological role was thought to be to protect cells from the harmful effect of nitroalkanes9. However, no functional genetic analysis of fungal NMO genes has previously been carried out. Marroquin-Guzman and co-workers created null mutants in two of the five NMO-encoding genes of M. oryzae, NMO2 and NMO4, and found that mutants lacking NMO2 are impaired when grown on nitrate or nitrite6. Both of these nitrogen sources can produce reactive nitrogen species (RNS) as a byproduct of their metabolism, which in turn generate nitroalkanes and other nitrated lipid species10. The authors reasoned that Nmo2 may play a role in mitigating RNS-induced lipid damage during growth of M. oryzae. Consistent with this idea, they found that the Δnmo2 strain is highly sensitive to nitric oxide or hydrogen peroxide, two determinants of RNS production and, when grown on nitrate, the mutant shows increased cellular levels of nitrotyrosine and malondialdehyde, two markers of nitrooxidative stress and RNS-induced lipid damage, respectively. They also found that growth of the Δnmo2 mutant on nitrate could be rescued in the presence of the peroxynitrite scavenger MnTBAP chloride, strongly suggesting that Nmo2 protects M. oryzae against lipid damage caused by nitrooxidative stress during nitrate and nitrite metabolism, and is thus required for the safe utilization of these two nitrogen sources6.

Intriguingly, the expression of NMO2 is upregulated in M. oryzae during plant infection and loss of the gene abolishes infection of rice leaves6. While the Δnmo2 mutant is still able to form functional appressoria and penetrate rice cells, its biotrophic growth within the plant cells is dramatically reduced. Strikingly, rice cells infected by the Δnmo2 mutant (but not those infected by the wild-type strain) display characteristic hallmarks of the plant immune response: high levels of ROS, upregulation of defence-related genes and deposition of callose in the cell wall. To test whether increased accumulation of ROS in rice cells infected by the Δnmo2 mutant was responsible for elicitation of the host immune response, the authors treated spores of the mutant with the NADPH oxidase inhibitor diphenyliodonium (DPI) or the ROS scavenger, ascorbate. Both of these treatments resulted in mitigation of the plant defence response and allowed the Δnmo2 mutant to extend colonization to adjacent host cells6. NMO2 therefore provides a novel means by which the rice blast fungus can adapt to, but also combat, the oxidative environment of plant cells.

More than 20 years have passed since a genetic screen in M. oryzae identified two mutants affected both in the utilization of nitrogen sources and in fungal pathogenicity11. While this early report suggested a close link between fungal nitrogen metabolism and plant infection, the underlying mechanisms have remained elusive. By uncovering two key roles of fungal NMOs — protection from nitrooxidative stress during nitrate metabolism and suppression of the plant oxidative burst — the current work by Marroquin-Guzman and colleagues provides new insight into the interplay between fungal primary metabolism and host manipulation. It also highlights the importance of the trehalose-6-phosphate synthase sensor — which regulates NMO2 — as a monitor of glucose availability that controls both nitrogen source utilization and anti-oxidation in a very precise manner, enabling the fungus to rapidly adapt to the changing conditions of host cells12.

Importantly, the finding that M. oryzae uses a stress response enzyme to maintain cell redox balance in the host cell and prevent its defence response challenges our current view of secreted effectors as the main mechanism by which pathogens manipulate host immune signalling8. This study is likely to inspire further research into the links between primary fungal metabolism and host infection. However, important questions also emerge. How does fungal NMO suppress or neutralize the plant oxidative burst? Does NMO-mediated protection from lipid nitrooxidative damage prevent amplification of host ROS through an unknown mechanism? Future work will help to address these issues, and reveal whether other filamentous pathogens have similarly recruited NMOs for modulating host immunity and increasing their infectious potential.

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Correspondence to Antonio Di Pietro or Nicholas J. Talbot.

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Di Pietro, A., Talbot, N. Fungal pathogenesis: Combatting the oxidative burst. Nat Microbiol 2, 17095 (2017). https://doi.org/10.1038/nmicrobiol.2017.95

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